1. Field of the Invention
The present invention relates generally to the fields of molecular biology, radiation oncology and cancer therapy. More specifically, the present invention relates to the finding that a combination of molecular chemotherapy and radiation therapy enhances therapeutic effects against cancer.
2. Description of the Related Art
Clinical applications of cancer gene therapy have had limited success due to a variety of factors, including ineffective therapeutic gene delivery in situ. The physiologic milieu of the target tumor may have deleterious effects on the delivery of therapeutic genes. This limitation may be disease specific, and variable depending on the specific tumor type and tumor location. Most clinical gene therapy trials thus far have utilized compartmental models of malignant disease (1, 2). In this regard, thoracic malignancies and intra-abdominal carcinomatosis represent common body compartmentalized diseases that have been explored in an experimental therapeutic context. Attempts to address the issue of achieving viral vector delivery to cancer cells in the face of a physiologic infection medium of pleural fluid or abdominal ascites have been examined (3, 4). Yang et al. demonstrated retroviral transduction of pancreatic cancer cells in the presence of human ascites, similar to the results obtained in culture medium (3). Batra et al. reported significant inhibition of retroviral transduction of mesothelioma cells in the presence of malignant pleural fluid, specifically the chondroitin sulfate proteoglycan fraction (4).
Radiotherapy combined with the radiosensitizing chemotherapeutic drug 5-fluorouracil (5-FU) has been studied as a therapeutic modality in many human tumor types (5). Systemic toxicity limits the amount of 5-FU that can be administered for many clinical anti-cancer applications (6, 7). Radiation therapy and gene therapy have the potential to be combined to enhance effectiveness of cancer therapy without enhancing dose limiting toxicity. To this end, reports have investigated this interaction (8). These include: TNF_ under the control of a radiation inducible promoter (9, 10), conversion of prodrugs to toxic metabolites that are also radiosensitizers (11-15), p53 mediated radiosensitization (16, 17) and the genetic induction of membrane receptors that can be targeted with radiolabeled peptides (18-21).
With respect to enzymatic conversion of nontoxic prodrugs into radiation sensitizing agents, the genes for bacterial and yeast cytosine deaminase (CD) have been cloned and studied (22, 23, 40). Cytosine deaminase converts a nontoxic prodrug 5-fluorocytosine (5-FC) into 5-FU. The cytosine deaminase gene has been used in gene therapy strategies to mediate intracellular conversion of 5-FC to 5-FU, and has been shown to be effective in animal tumor models of human colon carcinoma (24). Human colon cancer cells that have been stably transduced to express the cytosine deaminase gene have been shown to be radiosensitized by the addition of 5-FC in vitro and in vivo (13). Adenoviral vectors have been used to achieve efficient gene delivery in a variety of tissues in vitro and in vivo. Adenoviral vectors encoding the cytosine deaminase gene have been described (25, 26).
Presently available assays for determining intratumoral 5-FU concentration are problematic. They require the removal of a tumor, the homogenization of that tumor and the collection of the cellular lysate in order to directly measure 5-FU concentration, usually by high-pressure liquid chromatography. No noninvasive method of detection existed, which could allow for continuous in vivo monitoring of 5-FU production.
In the context of multiple administrations of adenoviral vectors, the host immunologic response, with generation of neutralizing anti-adenovirus antibodies and cytotoxic T cells, is thought to limit the potential effectiveness of secondary administration of adenoviral vectors. A means to overcome this problem may be to improve the effectiveness of infection of the initial viral challenge, i.e., to enhance the transduction efficiency of the adenoviral vector for the target cells at the initial adenoviral administration. This goal may be achieved by utilizing a ligand to a cellular receptor overexpressed in the target carcinoma cells to redirect adenovirus vector binding.
Primary central nervous system (CNS) tumors, arising in both the brain and spinal cord, are the leading cause of cancer-related deaths in children less than 15 years of age (42-44). They are the most common solid neoplasia in children with an estimated incidence of 3.77 newly diagnosed pediatric patients per 100,000 children at risk each year in the US (45). Despite aggressive treatment with radiation and/or chemotherapy, children with intrinsic brainstem gliomas and high-grade astrocytomas rarely survive more than a few years from diagnosis (46-49). The long-term sequelae of radiation are significant, especially in very young children, militating against its use as standard therapy in children less than 36 months old (50-52). In this context, gene therapy offers a promising approach for pediatric brain tumors.
The main factor currently limiting the clinical potential of gene therapy is the poor level of in situ tumor cell transduction achievable by existing gene transfer vectors (53). Of these, adenovirus (Ad) is particularly attractive due to its well-characterized mechanism of cellular entry, and its propensity to efficiently infect a wide variety of cell types within the CNS (54-56). This is presumably due to their expression of the cellular receptors necessary for efficient Ad entry, the coxsackie-adenovirus receptor (CAR) and xcex1v integrins (57, 58). Ad vectors have shown utility in several animal models of glioma (59, 60) and are currently being investigated in at least three separate clinical trials in the US in adult patients with malignant glioblastomas. Yet as all three employ direct intratumoral or intracavitary injection, expression of Ad receptors on these tumors will likely determine the overall success of these and future Ad cancer gene therapy trials. Two of these trials are investigating replication-defective Ad as a vector for enzyme/prodrug therapy using herpes simplex virus thymidine kinase/gancyclovir (HSV-tk/GCV, 61). While HSV-tk/GCV enzyme/prodrug therapy is promising for malignant gliomas, several alternatives have been described (reviewed in 62).
The third trial involves a replication-competent Ad lacking an exogenous transgene (ONYX-015) (76) and is being conducted in adult patients. ONYX-015 harbors an E1B-55K gene deletion that permits the selective replication in and lysis of cells with mutations in the gene encoding p53 (77). Replication-competent viruses such as Ad have distinct advantages over non-replicative viruses in cancer gene therapy (reviewed in 78, 79). First, Ad replication in tumor cells results in cell lysis (lytic infection) and hence tumor destruction (viral oncolysis). Second, lateral spread of progeny Ad virions within the productively infected tumor mass dramatically increases exogenous transgene expression compared with replication-defective vectors (80).
The prior art is deficient in the lack of effective means of treating of human cancers by chemotherapy combined with radiation therapy to produce enhanced therapeutic effects against cancer and reduced normal tissue toxicity. In addition, the prior art is deficient in the lack of effective means of redirecting adenovirus vector binding via a cellular receptor to improve the effectiveness of gene therapy. Furthermore, the prior art is deficient in the lack of a noninvasive method for continuously monitoring therapeutic transgene expression in tumors therefore improving the gene therapy. The present invention fulfills this long-standing need and desire in the art.
The present invention is directed to a method of transfecting established tumors in vivo with an adenovirus encoding the cytosine deaminase gene, administration of systemic 5-FC, and radiation therapy, (e.g., external beam or brachytherapy) of the tumor. This method results in tumor regression and prolonged tumor growth inhibition compared to control treatments with molecular chemotherapy or radiation therapy alone. Also disclosed is an adenoviral-conjugate mechanism to circumvent current limitations of cancer gene therapy to solid gastrointestinal malignancies.
Specifically, the present invention utilizes an adenoviral vector under the control of a cytomegalovirus promoter (AdCMVCD) encoding cytosine deaminase in combination with 5-FC and single fraction radiotherapy to demonstrate enhanced cytotoxicity to WiDr human colon carcinoma cells in vitro. The present invention also demonstrates such gene therapy/prodrug treatment strategy employing a fractionated radiation dosing schema in animal models of WiDr human colon carcinoma and SK-ChA-1 human cholangiocarcinoma. A prolonged WiDr tumor regrowth delay was obtained with AdCMVCD infection in combination with systemic delivery of 5-FC and fractionated external beam radiation therapy compared to control animals treated without radiation, without 5-FC, or without AdCMVCD. The present invention further discloses redirection of adenovirus vector (AdCMVCD) binding via a ligand to a cellular receptor, e.g., the basic fibroblast growth factor (FGF2) receptor, to improve the effectiveness of gene therapy in combination with 5-FC treatment and radiation therapy.
Clinical applications for cancer gene therapy are limited by the inability to genetically modify a majority of tumor cells to achieve a therapeutic effect. In this regard, the enzyme/prodrug strategy consisting of cytosine deaminase/5-fluorocytosine (CD/5-FC) relies on diffusion of the cytotoxic enzymatic product 5-FU to kill non-transduced tumor cells. Methods to increase solid tumor transduction in situ may augment therapeutic gene expression and response to therapy. To this end, gene delivery was improved via vector binding to molecules expressed on the cells of tumors. Fibroblast growth factor (FGF) receptors are overexpressed in a majority of pancreatic carcinomas, but poorly characterized in cholangiocarcinoma. Targeted adenovirus via basic fibroblast growth factor (FGF2) to the fibroblast growth factor receptor was used as a vehicle for the delivery of cytosine deaminase to hepatobiliary tumor cells for combination molecular chemotherapy and radiation therapy studies.
FGF2 redirected adenoviral delivery of firefly luciferase gene (AdCMVLuc) expression was evaluated in vitro. Transduction efficiencies using adenoviral delivered E. coli xcex2-galactosidase gene (AdCMVLacZ) expression also were determined. The methodology to redirect adenoviral gene delivery employed the Fab fragment of a neutralizing anti-adenoviral knob monoclonal antibody which ablates native adenoviral tropism, conjugated to FGF2 ligand which provides for FGF receptor binding. An adenoviral vector encoding the cytosine deaminase gene (AdCMVCD), in combination with 5-FC and the Fab-FGF2 conjugate, was used to evaluate differential cytosine deaminase protein expression by Western blotting of transfected cell lines and enzymatic activity by increased conversion of 3H-5-FC into 3H-5-FU. Proliferation assays were performed to correlate differential production of 5-FU with increased cytotoxicity in selected pancreatic and cholangiocarcinoma cell lines. In vivo studies utilizing AdCMVCD, the Fabxe2x80x2-FGF2 conjugate, 5-FC administration, and a single 5 Gy dose of external beam radiation to the tumor in nude mice were performed to evaluate the anti-tumor efficacy of AdCMVCD+Fabxe2x80x2-FGF2, compared to AdCMVCD alone, in established subcutaneous BXPC-3 pancreatic tumors.
In target cells, FGF2 retargeted AdCMVLuc resulted in enhanced (10-100-fold) levels of firefly luciferase expression relative to AdCMVLuc infection alone. X-gal staining for xcex2-galactosidase expression revealed an enhanced transduction frequency mediated by Fab-FGF2 redirected AdCMVLacZ compared to AdCMVLacZ infection alone. Fab-FGF2 redirection of AdCMVCD resulted in increased cellular expression of cytosine deaminase and production of 5-FU, and enhanced cellular cytotoxicity at low viral multiplicities of infection, compared to the levels obtained with AdCMVCD alone. In BXPC-3 tumor-bearing animals treated with AdCMVCD+Fabxe2x80x2-FGF2, 5-FC, and radiotherapy, the time to tumor size doubling was extended compared to AdCMVCD, 5-FC, and radiotherapy alone.
These results indicate that native adenoviral tropism can be redirected using ligands to cell surface receptors. In addition, transduction efficiencies and expression of genes introduced via this heterologous pathway are significantly enhanced compared to native adenovirus transduction alone. These findings suggest improved gene expression may be achieved via this adenoviral-conjugate mechanism to circumvent current limitations of cancer gene therapy to solid gastrointestinal malignancies.
The present invention is further directed to a noninvasive method for monitoring the continuous conversion of 5-fluorocytosine to 5-fluorouracil via magnetic resonance spectroscopy (MRS). Magnetic resonance spectroscopy allows for monitoring this prodrug activation therapy through the following: the identification of tumor and normal tissue sites of production or accumulation of 5-fluorouracil, the discrimination of both 5-fluorocytosine clearance/5-fluorouracil production, the determination of the residence time of 5-fluorouracil, the production of metabolites of the active drug, along with the determination of the elimination kinetics of 5-fluorouracil from tumor and normal organs. The information that magnetic resonance spectroscopy can provide about the pharmacokinetics of these agents can help develop procedures to maximize the effectiveness of this therapy with the potential to maximize tumor regression.
In one embodiment of the present invention, there is provided a method of treating an individual having a solid tumor, comprising the steps of treating the individual with an adenovirus encoding a cytosine deaminase gene; administering 5-fluorocytosine to the individual; and treating the individual with radiation therapy.
In another embodiment of the present invention, there is provided a method of treating an individual having a cancer, comprising the steps of combining a ligand that binds to a tumor cellular receptor and an adenoviral vector encoding a cytosine deaminase gene to form a complex; treating the individual with the complex; administering 5-fluorocytosine to the individual; and treating the individual with external beam irradiation.
In still another embodiment of the present invention, there is provided a method of monitoring continuous conversion of 5-fluorocytosine to 5-fluorouracil in a tumor, wherein the tumor is treated with multiple doses of 5-fluorocytosine and multiple doses of adenovirus encoding a cytosine deaminase gene, comprising the steps of placing the treated tumor in a magnet; and evaluating the presence of 5-fluorocytosine and 5-fluorouracil by magnetic resonance spectroscopy over a course of time, wherein less amount of 5-fluorocytosine and more amount of 5-fluorouracil indicates increased conversion of 5-fluorocytosine to 5-fluorouracil. Preferably, the tumor is further treated with radiation.
In still yet another embodiment of the present invention, there is provided a method of monitoring continuous conversion of 5-fluorocytosine to 5-fluorouracil in a tumor, wherein the tumor is treated with multiple doses of 5-fluorocytosine and multiple doses of cytosine deaminase gene encoding adenovirus targeted by a ligand to a tumor cellular receptor, comprising the steps of placing the treated tumor in a magnet; and evaluating the presence of 5-fluorocytosine and 5-fluorouracil by magnetic resonance spectroscopy over a course of time, wherein less amount of 5-fluorocytosine and more amount of 5-fluorouracil indicates increased conversion of 5-fluorocytosine to 5-fluorouracil. Preferably, the tumor is further treated with radiation.
The instant invention also provides an adenovirus which selectively replicates in tumor cells and encodes a cytosine deaminase gene. Preferably, the adenovirus has a complete E1A gene but lacks an E1B gene and selectively replicates in cells with a defective p53 pathway. AdE1ACD is a representative example of such an adenovirus.
The instant invention is further directed to a method of treating an individual having a solid tumor with a selectively replicating adenovirus encoding cytosine deaminase comprising the steps of infecting the individual with the adenovirus and administering 5-fluorocytosine followed by radiation therapy.
A further embodiment of the instant invention is directed to an adenovirus which coexpresses cytosine deaminase and uracil phosphoribosyltransferase preferably as a fusion protein as is the case with AdCDUPRT.
The present invention also includes a method of treating an individual having a solid tumor by administering an adenovirus coexpressing cytosine deaminase and uracil phosphoribosyltransferase and administering 5-fluorocytosine followed by radiation therapy.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.